Recoveries of Phenylalanine from Two Sets of Dried-Blood-Spot Reference Materials: Prediction from Hematocrit, Spot Volume, and Paper Matrix

Dried blood spots (DBSs) are used to screen newborns for phenylketonuria and other aminoacidopathies. The calibrators for this testing are usually DBSs with values for Phe. Two DBS reference materials have been prepared, the European Working Standard for Phe (EWS-Phe-01) (1) and the amino acid reference material (AARM) from the CDC (2). The two reference materials are not interchangeable because they differ in blood hematocrit, blood-spot size, and filter paper, each of which (3)(4)(5)(6) affects analyte recovery. We measured quantitatively the effects of these differences on analyte recovery from DBSs and used results from our measurements to predict expected Phe recoveries from tandem analyses of the two sets of materials. In EWS-Phe-01 (1)(7), human blood with a 50.5% hematocrit and intact red cells was divided into five portions for enrichment with 0, 20, 40, 80, and 120 mg Phe/L blood (0, 120, 240, 480, and 720 μmol/L blood). The liquid added during enrichment (7) was sufficient to reduce the hematocrit to 50.1%. The Phe-enriched blood portions were dispensed in 35-μL aliquots (7) onto Schleicher & Schuell (S&S) Grade 2992 (lot no. 121576) filter paper (1). The AARM was prepared (2) by dividing human blood with a 57% hematocrit and intact red cells into six portions for enrichment with pure amino acids to cover the usual analytic ranges of Phe, Tyr, Leu, Met, and Val. The Phe enrichments were 0, 40, 80, 120, 160, and 200 mg Phe/L blood (0, 240, 480, 720, 960, and 1200 μmol/L blood). The liquid added during enrichment was sufficient to reduce the hematocrit to 53%. The whole-blood pools were dispensed in 100-μL portions onto S&S Grade 903 (lot no. W941) filter paper with dashed-line 13-mm printed circles (2). To examine the effect of blood hematocrit …